Mapping paratopes of nanobodies using native mass spectrometry and ultraviolet photodissociation

Macias, Luis A.; Wang, Xun; Davies, Bryan William; Brodbelt, Jennifer S.

doi:10.1039/d2sc01536f

Cited by 5 publications

(1 citation statement)

References 61 publications

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“…A more recent study published by Macias et al applied ultraviolet photo-dissociation (UVPD) activation to fragment three different nanobody-antigen pairs. 54 This experiment was conducted under native MS conditions (nMS) with the main purpose of inferring structural insights onto the quaternary structure of nanobody-antigen pairs from their corresponding UVPD fragmentation spectra. In this context, the fragmentation of the apo-, and holo-forms of the nanobodies were compared to detect significant fragmentation differences upon antigen binding.…”

Section: Introductionmentioning

confidence: 99%

Development of fine-tuned top-down mass spectrometry strategies in the chromatographic time scale (LC-TD-MS) for the complete characterization of an anti EGFR single domain antibody-drug conjugate (sdADC)

Benazza,

Papadakos,

Thom

et al. 2024

Preprint

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Even though mAbs have attracted the biggest interest in the development of therapeutic proteins, next generation of therapeutics such as single-domain antibodies (sdAb) are propelling an increasing attention as new alternatives with appealing applications in different clinical areas. These constructs are small therapeutic proteins formed by a variable domain of the heavy chain of an antibody with multiple therapeutic and production benefits compared to their mAb counterparts. These proteins can be subjected to different bioconjugation processes to form single-domain antibody-drug conjugates (sdADC) and hence increase their therapeutic potency, and, akin to other therapeutic proteins, nanobodies and related products require dedicated analytical strategies to fully characterize their primary structure prior to their release to the market. In this study we report for the first time on the complete sequence characterization of a conjugated anti-EGFR 15 kDa sdADC by using cutting-edge top-down mass spectrometry strategies in combination with liquid chromatography (LC-TD-MS). Mass analysis revealed a highly homogeneous sample with one conjugated molecule. Subsequently, the reduced sdADC was submitted to different fragmentation techniques namely higher-energy collisional dissociation (HCD), electron-transfer dissociation (ETD), and ultraviolet photo-dissociation (UVPD) allowing to unambiguously asses the conjugation site with 16 diagnostic fragment ions and more than 85% of global sequence coverage. The sequence coverage of the non-reduced protein was significantly lower (around 33%), however the thorough analysis of the fragmentation spectra and the inclusion of the internal fragments corroborated the presence of the intra-molecular disulfide bridge along with the localization of the conjugation site. Altogether, our results pinpoint the complementary of the different fragmentation techniques to provide a thorough analytical characterization of sdAbs-formats even in the chromatographic time-scale, which allows a high-throughput and streamlined analysis of this kind of therapeutic proteins facilitating the implementation on dedicated R&D laboratories.

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Section: Introductionmentioning

confidence: 99%

Development of fine-tuned top-down mass spectrometry strategies in the chromatographic time scale (LC-TD-MS) for the complete characterization of an anti EGFR single domain antibody-drug conjugate (sdADC)

Benazza,

Papadakos,

Thom

et al. 2024

Preprint

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Analysis for disulfide bonds in polypeptide sequences based on ultraviolet photodissociation combined with linear ion trap mass spectrometry

He-Yi,

Di,

et al. 2023

Chinese Journal of Analytical Chemistry

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The role of venom proteomics and single-domain antibodies for antivenoms: Progress in snake envenoming treatment

Machado Marinho,

Chapeaurouge,

Dutra

et al. 2024

Drug Discovery Today

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Mapping paratopes of nanobodies using native mass spectrometry and ultraviolet photodissociation

Abstract: Following immense growth and maturity of the field in the past decade, native mass spectrometry has garnered widespread adoption for the structural characterization of macromolecular complexes. Routine analysis of biotherapeutics...

Cited by 5 publications

References 61 publications

Development of fine-tuned top-down mass spectrometry strategies in the chromatographic time scale (LC-TD-MS) for the complete characterization of an anti EGFR single domain antibody-drug conjugate (sdADC)

Development of fine-tuned top-down mass spectrometry strategies in the chromatographic time scale (LC-TD-MS) for the complete characterization of an anti EGFR single domain antibody-drug conjugate (sdADC)

Analysis for disulfide bonds in polypeptide sequences based on ultraviolet photodissociation combined with linear ion trap mass spectrometry

The role of venom proteomics and single-domain antibodies for antivenoms: Progress in snake envenoming treatment

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